1. Cell Adhesion to Polymer Surfaces George Tulevski Colloids and Surface phenomena

2. Introduction Cell adhesion to polymer surfaces has obvious implications in the field of tissue engineering Facilitating cellular adhesion, growth and differentiation onto a surface can aid in wound healing and tissue growth a polymer can provide mechanical stability for the newly forming tissue understanding adhesion mechanisms can prevent bacterial cell adhesion and aid in infection control

3. Composition of biomaterial surfaces 1 Substrate - polymer surface Conditioning layer - adsorbed macromolecues (i.e. polysaccharides, proteins Cells - adhesion of cells to conditioned biosurface Needs to be a clear understanding of composition of the three components and how they interact

4. Surface Characterization 3 Use contact angle measurements to obtain surface energy of substrate surface energy of substrate, along with that of the protein, governs the protein adsorption, including which proteins adsorb and to what degree

5. Surface spectroscopy: General Method 4 The solid sample is irradiated with a primary beam of electrons, photons, ions or molecules The impact of this beam, then generates a secondary beam which is sent to the analyzer

6. Surface spectroscopy: Examples 4

7. XPS 4 X-ray photoelectron spectroscopy a photon of a monochromatic x-ray beam displaces an electron from an orbital of the atom E b = hv – E K – w The ejected electron’s kinetic energy, E k,, is measured and can be used to determine E b,, which is characteristic of the atom. Elemental composition, functionality, oxidation states and structure can be elucidated

8. SIMS and SEM 4 SIMS - secondary ion mass spectrometry the surface is bombarded with beams of 5 to 20 keV ions, such as Ar +, Cs +, N 2 + or O 2 + by an ion gun. The surface is stripped of its molecules and the resultant charged molecules are sent to the mass analyzer (i.e. quadropole, flight tube) SEM - scanning electron microscopy surface is scanned with a beam of energetic electrons the secondary beam is created and consists of backscattered electrons and secondary electrons the secondary beam is then sent to the analyzer and morphological and topographical information is obtained

9. Formation of the conditioning film 2 The interaction between proteins on the surface and the cells has an enormous impact on cell adhesion Adhesion of animal cells is mediated by adhesion receptor proteins in the cells membrane (the integrin family is primarily responsible for cell/foreign substance adhesion) Adhesion proteins bind to ligand proteins adsorbed to the surface (i.e. fibrinogen, von Willebrand, vitronectin and fibronectin) Adsorbing these proteins to a surface and maintaining their bio-activity can facilitate cell growth

10. Protein adsorption 3 Proteins are surfactants- they have hydrophobic and hydrophilic portions and adsorb to virtually any surface the degree of adsorption is governed by the interfacial free energy between the protein and substrate  G IF 1w2 =  G LW 1w2 +  G AB 1w2 A favorable interaction corresponds to a negative value of  G IF 1w2 this value can be obtained from tensiometric measurements such as contact angle or wicking

11. Vroman Effect Protein adsorption is essentially irreversible if no other proteins are present Proteins can replace themselves at the surface Different proteins have different affinities for a surface, proteins with a stronger affinity can displace proteins with a weaker affinity at the surface When proteins are allowed to adsorb to steady state, and isotherm is formed in which (1) an adsorption plateau occurs at high bulk concentrations (2) at lower concentrations, adsorption is linear until it reaches a plateau

12. Protein adsorption (van Oss 1991) 14 Maximum cell (absence of protein) and protein adhesion occurs at surface tension of 33 mJ/M 2 Despite this, minimal cell adhesion occurs at this surface tension since proteins adhere first, the hydrated surface of the adsorbed protein is hydrophilic in nature and results in minimal cell adhesion correlates well with previous data showing that cells adhere least to polymer surfaces of this energy in vivo (Baier 1984) 10

13. Protein adsorption and cell adhesion: (Busscher 1989) 6 This work displays that cell adhesion increases with increasing surface energy cell adhesion moves from poor to good at around  s = 55 mJ/m 2 higher surface energy facilitates cell growth (fibroblasts, smooth muscle cells, epithelial cells bulk measurements can elucidate macroscopic interactions, but there needs to be a correlation between surface chemistry and cell adhesion to develop control over the system. Conformational changes occur regularly upon adsorption of a protein and can alter bio-activity. This also effects cell adhesion and cannot be explained by energetics alone.

14. Microscopic correlation : (van Oss 1995) 14 The adsorption of H.S.A. onto glass is energetically disfavored despite this, the “elbow” can overcome the macroscopic repulsion and obtain a local point of attachment via a divalent metal cation (i.e. Ca 2+ ) electron acceptor interacting with an electron donor in the protein with little conformational changes Determined by adding EDTA and then observing detachment of the protein

15. Minimal Peptide Sequences One can circumvent problems with conformational changes in the protein by using minimal peptide sequences. The most influential sequence in many ligand proteins is the RGDS (arg-gly-asp-ser) sequence this sequence can be covalently linked to the surface and be used to bind to the adhesion receptor protein in the cells membrane and encourage adhesion Example: Brandley and Schnaar 1988 used (tyr-ala-val-gly-arg-gly-asp-ser) sequence and covalently immobilized it to a polyacrylamide gel surface the surface was coated with a density of 2 nmol peptide/cm 2 and supported long-term fibroblast growth tertiary structure must have some importance and a role in mediating cell adhesion.

16. Specific case: (Busscher 1992) 12 The author used e-PTFE vascular grafts that were luminally modified and implanted into rabbit carotid artery the interior was modified by ion etching and was followed by oxygen glow-discharge to render the surface highly hydrophobic the water contact angle went from 109 to 140-150 degrees for the modified surface the graft was implanted and then removed after one week and inspected with SEM the lumen had some platelet adhesion, but did not experience a cascade of platelet adhesion which would have led to clotting the outside, anastomic side, had some endothelial outgrowth, as desired the biomaterial succeeded on both fronts in providing a pathway for blood flow and tissue growth around the outside.

17. SEM images 12

18. Conclusions Four aspects to address: –Biomaterial substrate must be well characterized including its chemical composition, morphology and structure. –.A firm understanding of macromolecular film conditioning structure, conformation and interaction with the substrate. –Cell processes such as retention, adhesion, differentiation and growth should be quantitatively measured. –Short and long term effects should be evaluated –Future trend: ability to control formation of conditioning layer with selectivity and conformational control of adsorbed protein. Correlations between surface chemistry and protein conformation and how to modify the surface chemistry to obtain desired results